Submitted to: Analytical Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/16/2007
Publication Date: 6/15/2007
Citation: Wang, Z., Zhu, Y., Ding, S., He, F., Beier, R.C., Li, J., Jiang, H., Feng, C., Wan, Y., Zhang, S., Kai, Z., Yang, X., Shen, J. 2007. Development of a monoclonal antibody-based broad-specificity ELISA for fluroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds. Analytical Chemistry. 79:4471-4483. Interpretive Summary: Fluoroquinolones (FQs) are synthetic antibiotics derived from the quinolone nalidixic acid, which makes these antibiotics more effective against Gram-negative bacteria and also widens the quinolone spectrum of activity to include activity against Gram-positive bacteria. These antibiotics are used for treatment in livestock and poultry. We have developed antibodies that recognize a broad group of twelve FQ antibiotics. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once the antibody to a foreign substance is isolated, it can be used in a method to detect the presence of that foreign substance. The antibodies that we isolated may be used in a easy-to-use test to determine the levels of fluoroquinolones in food products. These tests would minimize the risk of FQ exposure during human consumption of animal products.
Technical Abstract: Development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR) and ofloxacin (OFL) were conjugated to bovine serum albumin for immunogens and ovalbumin for coating antigens. The Mab C-4A9-H1 raised against the CIP hapten exhibited high cross-reactivity (35 to 100%) with 12 of 14 FQs. After optimization, the generic antibodies were capable of detecting at least 12 FQs in a ciELISA below the maximum residue levels (MRLs) with good sensitivity at 50% binding inhibition (IC50). The specificity and cross-reactivity of Mab C-4A9-H1 is discussed in relation to the three-dimensional, computer-generated molecular models of the FQs. The quantitative structure-activity relationship (QSAR) between Mab C-4A9-H1 and various FQ drugs was obtained by comparative molecular field analysis (CoMFA). The obtained CoMFA model showed a high predictive ability with a cross-validation q**2 value of 0.866. Finally, the ciELISA developed was adapted for analysis of chicken muscle, chicken liver, honey, shrimp, and whole egg samples without a complicated purification process. These studies demonstrated that the recoveries were in the range of 60-93% for CIP, ENR, NOR, OFL, flumequine and danofloxacin in the five food matrices.